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2.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 1): 47-53, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30605125

RESUMO

Human endothelin is a 21-amino-acid polypeptide, constrained by two intra-chain disulfide bridges, that is made by endothelial cells. It is the most potent vasoconstrictor in the body and is crucially important in the regulation of blood pressure. It plays a major role in a host of medical conditions, including hypertension, diabetes, stroke and cancer. Endothelin was crystallized 28 years ago in the putative space group P6122, but the structure was never successfully solved by X-ray diffraction. Using X-ray diffraction data from 1992, the structure has now been solved. Assuming a unit cell belonging to space group P61 and a twin fraction of 0.28, a solution emerged with two, almost identical, closely associated molecules in the asymmetric unit. Although the data extended to beyond 1.8 Šresolution, a model containing 25 waters was refined to 1.85 Šresolution with an R of 0.216 and an Rfree of 0.284. The disulfide-constrained `core' of the molecule, amino-acid residues 1-15, has a main-chain conformation that is essentially the same as endothelin when bound to its receptor, but many side-chain rotamers are different. The carboxy-terminal `tail' comprising amino-acid residues 16-21 is extended as when receptor-bound, but it exhibits a different conformation with respect to the `core'. The dimer that comprises the asymmetric unit is maintained almost exclusively by hydrophobic interactions and may be stable in an aqueous medium.


Assuntos
Cristalografia por Raios X/estatística & dados numéricos , Endotelina-1/química , Peptídeos/química , Vasoconstritores/química , Água/química , Sequência de Aminoácidos , Pressão Sanguínea/fisiologia , Dissulfetos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Proteica , Multimerização Proteica
3.
Nat Struct Mol Biol ; 25(7): 570-576, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29967536

RESUMO

Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the 'core' binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the 'extended' binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs.


Assuntos
Angiotensina II/análogos & derivados , Receptor Tipo 2 de Angiotensina/química , Sítio Alostérico , Sequência de Aminoácidos , Angiotensina II/química , Angiotensina II/metabolismo , Cristalografia por Raios X , Endotelina-1/química , Endotelina-1/metabolismo , Humanos , Fragmentos de Imunoglobulinas , Cinética , Ligantes , Modelos Moleculares , Conformação Proteica , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Transdução de Sinais , Eletricidade Estática
4.
Sci Rep ; 7(1): 4956, 2017 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-28694457

RESUMO

Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac and renal diseases, and in the progression of tumour growth in cancer, but current diagnosis and treatment remain inadequate. Peptides derived from the 212 amino acid precursor preproendothelin-1 (ppET-1) may have utility as biomarkers, or cause biological effects that are unaffected by endothelin receptor antagonists. Here, we used specific immunoassays and LC-MS/MS to identify NT-proET-1 (ppET-1[18-50]), Endothelin-Like Domain Peptide (ELDP, ppET-1[93-166]) and CT-proET-1 (ppET-1[169-212]) in conditioned media from cultured endothelial cells. Synthesis of these peptides correlated with ET-1, and plasma ELDP and CT-proET-1 were elevated in patients with chronic heart failure. Clearance rates of NT-proET-1, ELDP and CT-proET-1 were determined after i.v. injection in anaesthetised rats. CT-proET-1 had the slowest systemic clearance, hence providing a biological basis for it being a better biomarker of ET-1 synthesis. ELDP contains the evolutionary conserved endothelin-like domain sequence, which potentially confers biological activity. On isolated arteries ELDP lacked direct vasoconstrictor effects. However, it enhanced ET-1 vasoconstriction and prolonged the increase in blood pressure in anaesthetised rats. ELDP may therefore contribute to disease pathogenesis by augmenting ET-1 responses.


Assuntos
Células Endoteliais/citologia , Endotelina-1/metabolismo , Insuficiência Cardíaca/diagnóstico , Fragmentos de Peptídeos/administração & dosagem , Precursores de Proteínas/química , Células A549 , Biomarcadores/sangue , Linhagem Celular , Cromatografia Líquida , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/metabolismo , Endotelina-1/química , Insuficiência Cardíaca/metabolismo , Humanos , Injeções Intravenosas , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacocinética , Espectrometria de Massas em Tandem
5.
J Magn Reson Imaging ; 46(2): 505-517, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28703413

RESUMO

PURPOSE: Stroke is the leading cause of adult disability worldwide. The absence of more effective interventions in the chronic stage-that most patients stand to benefit from-reflects uncertainty surrounding mechanisms that govern recovery. The present work investigated the effects of a novel treatment (selective cyclooxygenase-1, COX-1, inhibition) in a model of focal ischemia. MATERIALS AND METHODS: FR122047 (COX-1 inhibitor) was given beginning 7 days following stroke (cortical microinjection of endothelin-1) in 23 adult male rats. Longitudinal continuous-arterial-spin-labeling was performed prior to treatment (7 days), and repeated following treatment (21 days) on a 7T magnetic resonance imaging (MRI) system to estimate resting perfusion and reactivity to hypercapnia. These in vivo measurements were buttressed by immunohistochemistry. RESULTS: Stroke caused an increase in perilesional resting perfusion (peri-/contralesional perfusion ratio of 170 ± 10%) and perfusion responses to hypercapnia (180 ± 10%) at 7 days. At 21 days, placebo-administered rats showed normalized perilesional perfusion (100 ± 20%) but persistent hyperreactivity (190 ± 20%). Treated animals exhibited sustained perilesional hyperperfusion (180 ± 10%). Further, reactivity lateralization did not persist following treatment (peri- vs. contralesional reactivity: P = 0.002 at 7 vs. P = 0.2 at 21 days). Hemodynamic changes were accompanied by neuronal loss, increased endothelial density, and widespread microglial and astrocytic activation. Moreover, relative to controls, treated rats showed increased perilesional neuronal survival (22 ± 1% vs. 14.9 ± 0.8%, P = 0.02) and decreased microglia/macrophage recruitment (17 ± 1% vs. 20 ± 1%, P = 0.05). Finally, perilesional perfusion was correlated with neuronal survival (slope = 0.14 ± 0.05; R2 = 0.7, P = 0.03). CONCLUSION: These findings shed light on the role of COX-1 in chronic ischemic injury and suggest that delayed selective COX-1 inhibition exerts multiple beneficial effects on the neurogliovascular unit. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 4 J. MAGN. RESON. IMAGING 2017;46:505-517.


Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Isquemia/diagnóstico por imagem , Imagem por Ressonância Magnética , Proteínas de Membrana/antagonistas & inibidores , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/fisiopatologia , Animais , Ciclo-Oxigenase 1 , Modelos Animais de Doenças , Endotelina-1/química , Macrófagos/patologia , Masculino , Microglia/patologia , Neuroglia/patologia , Neurônios/patologia , Perfusão , Piperazinas/química , Ratos , Ratos Sprague-Dawley , Marcadores de Spin , Tiazóis/química
6.
Sci Rep ; 7(1): 3740, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28623260

RESUMO

Membrane proteins are key elements in cell-mediated processes. In particular, G protein-coupled receptors (GPCRs) have attracted increasing interest since they affect cellular signaling. Furthermore, mutations in GPCRs can cause acquired and inheritable diseases. Up to date, there still exist a number of GPCRs that has not been structurally and functionally analyzed due to difficulties in cell-based membrane protein production. A promising approach for membrane protein synthesis and analysis has emerged during the last years and is known as cell-free protein synthesis (CFPS). Here, we describe a simply portable method to synthesize GPCRs and analyze their ligand-binding properties without the requirement of additional supplements such as liposomes or nanodiscs. This method is based on eukaryotic cell lysates containing translocationally active endogenous endoplasmic reticulum-derived microsomes where the insertion of GPCRs into biologically active membranes is supported. In this study we present CFPS in combination with fast fluorescence-based screening methods to determine the localization, orientation and ligand-binding properties of the endothelin B (ET-B) receptor upon expression in an insect-based cell-free system. To determine the functionality of the cell-free synthesized ET-B receptor, we analyzed the binding of its ligand endothelin-1 (ET-1) in a qualitative fluorescence-based assay and in a quantitative radioligand binding assay.


Assuntos
Endotelina-1/metabolismo , Fluorescência , Receptor de Endotelina B/metabolismo , Transdução de Sinais , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Endotelina-1/química , Humanos , Receptor de Endotelina B/química
7.
Life Sci ; 179: 139-146, 2017 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-28483438

RESUMO

AIMS: Transplantation of a tissue engineered cardiac pacemaker (TECP) may represent a novel therapy for cardiac sinus node dysfunction. We previously reported that cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiac pacemaking cells after endothelin-1 treatment. We aimed to examine the feasibility of TECP fabricated from CPCs-derived pacemaking cells and vascularization of TECP fabricated from CPCs-derived pacemaking cells and endothelial progenitor cells (EPCs) in vitro and in vivo implantation. MAIN METHODS: TECP created using CPCs-derived pacemaking cells and vTECP created by mixing CPCs and EPCs in vitro were implanted into rat hearts. Sinus node damaged was induced by formaldehyde insult. KEY RESULTS: Spontaneous beating tissues, namely TECP, were obtained after seeding CPCs-derived pacemaking cells into Matrigel. ECG and epicardial multielectrode array (MEA) measurements confirmed implanted TECP have electrical activity. TECP implantation promoted individual survival in sinus node damage models (15/22 animals lived versus 0/17 control). vTECP fabricated by mixing the both EPCs and CPCs-derived pacemaking cells with Matrigel in equal proportions optimally formed pre-vascularization in vitro. The implantation of vTECP enhanced electrical activity in vivo, which may correlate with increased vascularization. PI3K-Akt-VEGF/VEGFR signaling was involved with vascular ingrowth in vTECP. SIGNIFICANCE: Our data supports the therapeutic potential of TECP fabricated with the CPCs-derived pacemaking cells for sinus node dysfunction. Vascularization by the addition of EPCs is an important factor to sustain viability of the TECP in vivo.


Assuntos
Colágeno , Células Progenitoras Endoteliais/citologia , Laminina , Proteoglicanas , Síndrome do Nó Sinusal/terapia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Modelos Animais de Doenças , Combinação de Medicamentos , Endotelina-1/química , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
8.
Biomed Res Int ; 2017: 9475074, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28299338

RESUMO

The compounds of Radix Paeoniae Rubra (RPR) were isolated and identified by bioassay-guided method, and antithrombotic effects and mechanism were investigated by the acute blood stasis rat model. The RPR extract was evaluated by APTT, TT, PT, and FIB assays in vitro. Results indicated that RPR extract exhibited the anticoagulant activity. In order to find active compounds, six compounds were isolated and identified, and four compounds, paeoniflorin (Pae), pentagalloylglucose (Pen), albiflorin (Ali), and protocatechuic acid (Pro), exhibited the anticoagulant activity in vitro. Therefore, the antithrombosis effects of RPR extract and four active compounds were investigated in vivo by measuring whole blood viscosity (WBV), plasma viscosity (PV), APTT, PT, TT, and FIB. Meanwhile, the levels of TXB2, 6-Keto-PGF1α , eNOS, and ET-1 were detected. Results suggested that RPR extract and four active compounds had the inhibition effect on thrombus formation, and the antithrombotic effects were associated with the regulation of vascular endothelium active substance, activating blood flow and anticoagulation effect.


Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Fibrinolíticos/uso terapêutico , Paeonia/química , Trombose/tratamento farmacológico , 6-Cetoprostaglandina F1 alfa/química , Animais , Anticoagulantes/química , Anticoagulantes/uso terapêutico , Bioensaio , Hidrocarbonetos Aromáticos com Pontes/química , Medicamentos de Ervas Chinesas/química , Endotelina-1/química , Feminino , Fibrinolíticos/química , Glucosídeos/química , Taninos Hidrolisáveis/química , Hidroxibenzoatos/química , Masculino , Monoterpenos/química , Óxido Nítrico Sintase Tipo III/metabolismo , Fitoterapia , Ratos , Ratos Sprague-Dawley , Tromboxano B2/química , Viscosidade
9.
Nature ; 537(7620): 363-368, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27595334

RESUMO

Endothelin, a 21-amino-acid peptide, participates in various physiological processes, such as regulation of vascular tone, humoral homeostasis, neural crest cell development and neurotransmission. Endothelin and its G-protein-coupled receptor are involved in the development of various diseases, such as pulmonary arterial hypertension, and thus are important therapeutic targets. Here we report crystal structures of human endothelin type B receptor in the ligand-free form and in complex with the endogenous agonist endothelin-1. The structures and mutation analysis reveal the mechanism for the isopeptide selectivity between endothelin-1 and -3. Transmembrane helices 1, 2, 6 and 7 move and envelop the entire endothelin peptide, in a virtually irreversible manner. The agonist-induced conformational changes are propagated to the receptor core and the cytoplasmic G-protein coupling interface, and probably induce conformational flexibility in TM6. A comparison with the M2 muscarinic receptor suggests a shared mechanism for signal transduction in class A G-protein-coupled receptors.


Assuntos
Endotelina-1/metabolismo , Receptor de Endotelina B/química , Receptor de Endotelina B/metabolismo , Regulação Alostérica , Sítio Alostérico , Membrana Celular/metabolismo , Cristalografia por Raios X , Endotelina-1/química , Endotelina-1/farmacologia , Endotelina-3/química , Endotelina-3/metabolismo , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/genética , Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Transdução de Sinais , Especificidade por Substrato
10.
Biochem J ; 473(12): 1651-62, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27288028

RESUMO

We introduce various, recently developed, generalized ensemble methods, which are useful to sample various molecular configurations emerging in the process of protein-protein or protein-ligand binding. The methods introduced here are those that have been or will be applied to biomolecular binding, where the biomolecules are treated as flexible molecules expressed by an all-atom model in an explicit solvent. Sampling produces an ensemble of conformations (snapshots) that are thermodynamically probable at room temperature. Then, projection of those conformations to an abstract low-dimensional space generates a free-energy landscape. As an example, we show a landscape of homo-dimer formation of an endothelin-1-like molecule computed using a generalized ensemble method. The lowest free-energy cluster at room temperature coincided precisely with the experimentally determined complex structure. Two minor clusters were also found in the landscape, which were largely different from the native complex form. Although those clusters were isolated at room temperature, with rising temperature a pathway emerged linking the lowest and second-lowest free-energy clusters, and a further temperature increment connected all the clusters. This exemplifies that the generalized ensemble method is a powerful tool for computing the free-energy landscape, by which one can discuss the thermodynamic stability of clusters and the temperature dependence of the cluster networks.


Assuntos
Proteínas/química , Proteínas/metabolismo , Endotelina-1/química , Endotelina-1/metabolismo , Modelos Biológicos , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Termodinâmica
11.
Analyst ; 140(24): 8092-6, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26548776

RESUMO

Endothelin-1 is a potent vasoconstrictive peptide that plays an important role in ex vivo lung perfusion. ET-1 expression levels are predictive of lung transplant outcomes and represent a valuable monitoring tool for surgeons; however, traditional techniques that measure [ET-1] are not suitable for the transplant setting. Herein, we demonstrate a new assay that rapidly measures ET-1 peptide levels in lung perfusate.


Assuntos
Técnicas de Química Analítica/métodos , Endotelina-1/química , Pulmão/patologia , Peptídeos/química , Bioensaio , Humanos , Pulmão/metabolismo , Transplante de Pulmão , Perfusão , Fatores de Tempo
12.
Am J Physiol Endocrinol Metab ; 307(8): E695-702, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25159325

RESUMO

Ocular ischemic microenvironment plays a critical role in the progression of diabetic retinopathy (DR). In this study, we investigated the effect of vitreous and aqueous obtained from proliferative DR patients on the function of CD34⁺ cells derived from healthy humans. Human CD34⁺ cells were incubated with vitreous or aqueous of subjects with PDR. After incubation, cell migration of CD34⁺ was evaluated with CXCL12. Intracellular levels of nitric oxide (NO) were measured with DAF-FM. Tube formation assay was used to evaluate the effect of treated CD34⁺ cells on in vitro angiogenesis. Angiogenic protein array and mass spectrometry (MS) were performed to ascertain the factors secreted by healthy nondiabetic CD34⁺ cells exposed to diabetic vitreous or aqueous. PDR vitreous/aqueous reduced migration of CD34⁺ cells (672.45 ± 42.1/736.75 ± 101.7 AFU; P < 0.01) and attenuated intracellular NO levels (182 ± 1.4/184.5 ± 6.3 AFU, P = 0.002). Pretreatment with PDR vitreous suppressed tube formation of human retinal endothelial cells (64 ± 1.6 vs. 80 ± 2.5). CD34⁺ exposed to PDR vitreous resulted in the increased expression of CXCL4 and serpin F1, whereas CD34⁺ exposed to PDR aqueous showed increased expression of CXCL4, serpin F1, and endothelin-1 (ET-1). MS analysis of CD34⁺ (exposed to PDR vitreous) expressed J56 gene segment, isoform 2 of SPARC-related modular calcium-binding protein 2, isoform 1 of uncharacterized protein c1 orf167, integrin α-M, and 40s ribosomal protein s21. Exposure of healthy nondiabetic CD34⁺ cells to PDR vitreous and aqueous resulted in decreased migration, reduced generation of NO, and altered paracrine secretory function. Our results suggest that the contribution of CD34⁺ cells to the aberrant neovascularization observed in PDR is driven more by the proangiogenic effects of the retinal cells rather than the influence of the vitreous.


Assuntos
Humor Aquoso/metabolismo , Retinopatia Diabética/metabolismo , Endotelina-1/metabolismo , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Fator Plaquetário 4/metabolismo , Serpinas/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Corpo Vítreo/metabolismo , Adulto , Células-Tronco Adultas/citologia , Células-Tronco Adultas/imunologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Idoso , Antígenos CD34/metabolismo , Humor Aquoso/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Células Cultivadas , Quimiotaxia , Retinopatia Diabética/imunologia , Retinopatia Diabética/patologia , Endotelina-1/agonistas , Endotelina-1/química , Proteínas do Olho/agonistas , Proteínas do Olho/química , Humanos , Pessoa de Meia-Idade , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fatores de Crescimento Neural/agonistas , Fatores de Crescimento Neural/química , Óxido Nítrico/metabolismo , Mapeamento de Peptídeos , Fator Plaquetário 4/agonistas , Fator Plaquetário 4/química , Retina/imunologia , Retina/metabolismo , Retina/patologia , Serpinas/agonistas , Serpinas/química , Vitreorretinopatia Proliferativa/imunologia , Vitreorretinopatia Proliferativa/patologia , Corpo Vítreo/imunologia
13.
Gene ; 548(2): 256-62, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25042451

RESUMO

Dilated cardiomyopathy (DCM) is a myocardial disease of unknown etiology with left ventricular dilatation and impaired myocardial contractility leading to heart failure. It is considered to be a multifactorial disorder with the interplay of both genetic and environmental factors. One of the possible genes implicated in DCM is endothelin 1 (EDN1). The genetic variants of EDN1 may be involved in the pathophysiology of DCM hence the entire EDN1 gene was screened to examine for the possible genotypic associations with DCM. A total of 115 DCM patients and 250 control subjects were included in the present study. PCR based SSCP analysis was carried out followed by commercial sequencing. Screening of EDN1 revealed two common and two rare polymorphisms. Allelic and genotypic frequencies were estimated in patient and control groups by appropriate statistical tests. The heterozygotes of insertion variation (+138A) were found to exhibit four-fold increase risk to DCM (OR=4.12, 95% CI 2.10-8.08; p=0.0001). The two rare variants (G>A transition (rs150035515) at c.90 and C>T transition (rs149399492) at c.119) observed in the present study were found to be unique in DCM. The secondary mRNA structures of these variations were found to have less free energy than wild type. The haplotype analysis revealed 4A-T to be risk haplotype for DCM (OR 5.90, 95% CI 2.29-15.25, p=0.0001). In conclusion, EDN1 polymorphisms (+138A, A30A, T40I) appear to play a significant role in the pathogenesis of DCM, as they influence the stability of protein. The increased EDN1 production may lead to constriction of coronary arteries, reducing coronary blood flow which may in turn increase the load on left ventricle, impairing contractility of the heart resulting in a DCM phenotype, an end stage of heart failure.


Assuntos
Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/fisiopatologia , Endotelina-1/genética , Endotelina-1/metabolismo , Adolescente , Adulto , Cardiomiopatia Dilatada/patologia , Endotelina-1/química , Éxons , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênese Insercional , Fenótipo , Mutação Puntual , Estabilidade Proteica , Adulto Jovem
14.
J Biomed Nanotechnol ; 9(8): 1345-53, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23926800

RESUMO

Endarterectomy and bypass surgery to treat renal artery stenosis are increasingly shunned these days due to high risks of complications during and after the surgery. Striving to find a sound alternative solution, we pioneered the construction of a tissue engineered renovascular graft that could immediately restore the normal blood flow to kidneys and sustain renal functions without suffering restenosis after the surgery. A highly porous scaffold was first constructed by electrospinning polycaprolactone, poliglecaprone, gelatin and elastin, giving the vast majority of non-woven fibers in the scaffold a diameter below 1200 nm. To recapitulate the anatomical and functional signatures of renal arteries, a bi-layer vasculature comprising a smooth muscle layer topped by an endothelial layer was built on the scaffold. The vasculature witnessed a sustained proliferation for up to 10 days in vitro and robustly secreted prostacyclin and endothelin-1, evidencing that the vasculature was functionally comparable to native renal arteries. After 30 days as a renovascular graft in mice, the luminal diameter of the graft remained clear without a restenosis and an increased confluence of the endothelial layer was observed. The tensile test confirmed that the renovascular graft was mechanically superior to native renal arteries and retained this advantage within 30 days in vivo. Also, this renovascular graft sustained renal functions as evidenced by normal levels of serum creatinine, urine creatinine and serum urea nitrogen and the lack of edema in the kidney cortex. These results demonstrate that this renovascular graft holds a great therapeutic promise for renal artery stenosis.


Assuntos
Prótese Vascular , Células Endoteliais/citologia , Miócitos de Músculo Liso/citologia , Polímeros/síntese química , Proteínas/química , Obstrução da Artéria Renal/cirurgia , Animais , Células Cultivadas , Creatinina/química , Creatinina/farmacologia , Dioxanos/química , Elastina/química , Elastina/farmacologia , Endarterectomia/instrumentação , Endarterectomia/métodos , Células Endoteliais/fisiologia , Células Endoteliais/transplante , Endotelina-1/química , Endotelina-1/farmacologia , Feminino , Gelatina/química , Gelatina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Miócitos de Músculo Liso/fisiologia , Miócitos de Músculo Liso/transplante , Poliésteres/química , Poliésteres/farmacologia , Polímeros/química , Artéria Renal/cirurgia , Tecidos Suporte/química
15.
Biochim Biophys Acta ; 1828(9): 2182-92, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23747296

RESUMO

The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured in association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications.


Assuntos
Endotelina-1/química , Lipossomos/química , Receptor de Endotelina A/química , Receptor de Endotelina B/química , Sistema Livre de Células/metabolismo , Detergentes/química , Endotelina-1/metabolismo , Expressão Gênica , Humanos , Cinética , Nanoestruturas/química , Ligação Proteica , Dobramento de Proteína , Receptor de Endotelina A/biossíntese , Receptor de Endotelina A/genética , Receptor de Endotelina B/biossíntese , Receptor de Endotelina B/genética
16.
J Vet Intern Med ; 27(4): 982-4, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23750948

RESUMO

BACKGROUND: There is a need for biomarkers for diagnosis, therapeutic monitoring, and prognosis for asthma in cats. Endothelin-1 (ET-1) is implicated in the pathogenesis of inflammatory airway diseases in other species but not the cat. OBJECTIVE: To conduct a prospective experimental study to show that experimentally asthmatic cats, but not control cats without airway inflammation, would have increased concentrations of ET in BALF. ANIMALS: Eleven healthy, adult research cats. METHODS: Prospective experimental study. Six healthy cats without airway inflammation were used as controls. Asthma was induced using Bermuda grass allergen (BGA) in 5 cats. Collection of BALF for total nucleated cell and differential counts was performed. The concentration of ET-1 in cell-free BALF samples was determined. Data were analyzed using a Mann-Whitney U-test with P < .05 considered significant. RESULTS: The median [range] BALF total cell numbers, eosinophil numbers, and eosinophil percentages were significantly higher in the cats following experimental induction of asthma (1,870 cells/µL [1,450-3,440], 711 cells/µL [356-1,686] and 38% [20-49]) compared to baseline control parameters (462 cells/µL [239-780], 18 cells/µL [18-62] and 3.5% [0-8]) (P < .01). The median [range] BALF ET concentration was also significantly higher after induction of asthma (1.393 fmol/mL[0.977-2.247]) compared to healthy control cats (0.83250 fmol/mL [0.625-1.038]) (P = .012). CONCLUSIONS AND CLINICAL IMPORTANCE: This study suggests that BAL ET-1 concentration can be used to differentiate normal cats from those with experimentally induced asthma. If the same holds true for cats with naturally developing asthma, BAL ET-1 may prove a useful diagnostic biomarker for asthma.


Assuntos
Asma/veterinária , Líquido da Lavagem Broncoalveolar/química , Doenças do Gato/induzido quimicamente , Endotelina-1/metabolismo , Alérgenos/imunologia , Alérgenos/toxicidade , Animais , Asma/induzido quimicamente , Asma/metabolismo , Doenças do Gato/metabolismo , Gatos , Cynodon/imunologia , Endotelina-1/química
17.
J Chem Phys ; 138(18): 184106, 2013 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-23676028

RESUMO

We propose a novel generalized ensemble method, a virtual-system coupled multicanonical molecular dynamics (V-McMD), to enhance conformational sampling of biomolecules expressed by an all-atom model in an explicit solvent. In this method, a virtual system, of which physical quantities can be set arbitrarily, is coupled with the biomolecular system, which is the target to be studied. This method was applied to a system of an Endothelin-1 derivative, KR-CSH-ET1, known to form an antisymmetric homodimer at room temperature. V-McMD was performed starting from a configuration in which two KR-CSH-ET1 molecules were mutually distant in an explicit solvent. The lowest free-energy state (the most thermally stable state) at room temperature coincides with the experimentally determined native complex structure. This state was separated to other non-native minor clusters by a free-energy barrier, although the barrier disappeared with elevated temperature. V-McMD produced a canonical ensemble faster than a conventional McMD method.


Assuntos
Endotelina-1/química , Simulação de Dinâmica Molecular , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Solventes/química
18.
J Pept Sci ; 19(4): 257-62, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23436727

RESUMO

The dimerization of the G protein-coupled receptors for endothelin-1 (ET-1), endothelin A receptor (ETA) and endolethin B receptor (ETB), is well established. However, the signaling consequences of the homodimerization and heterodimerization of ETA and ETB is not well understood. Here, we demonstrate that peptides derived from the C-termini of these receptors regulate the signaling capacity of ET-1. The C-termini of the ETA and ETB receptors are believed to consist of three α-helices, which may serve as points of interaction between the receptors. The third α-helix in the C-terminus is of particular interest because of its amphipathic nature. In a cell line expressing only the ETA receptor, expression of residues Y430-S442, representing the third helix of the ETB C-terminus, leads to a dramatic increase in the signaling induced by ET-1. In contrast, in a cell line containing only ETB , Y430-S442 has an antagonistic effect, slightly reducing the ET-1 induced signal. Computational docking results suggest that the α-helical ETB -derived peptide binds to the second and third intracellular loops of the ETA receptor consistent with the alteration of its signaling capacity. Our results described here provide important insight into ETA /ETB receptor interactions and possibly a new approach to regulate specific G protein-coupled receptor signal transmission.


Assuntos
Endotelina-1 , Receptor de Endotelina A , Receptor de Endotelina B , Transdução de Sinais/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Endotelina-1/química , Endotelina-1/genética , Endotelina-1/metabolismo , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Receptor de Endotelina A/química , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/química , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo
19.
Biol Reprod ; 87(6): 134, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23034156

RESUMO

Preeclampsia affects 5%-8% of pregnancies and is the leading worldwide cause of maternal and fetal morbidity and mortality. Preeclampsia is associated with shallow trophoblast invasion and inadequate spiral artery remodeling, which are widely believed to lead to placental hypoxia, the putative culprit initiating the cascade of events that ultimately results in the maternal manifestations of the disease. Despite extensive research, however, the pathophysiology of this disease remains poorly understood, no effective prevention exists, and treatment is limited to symptomatic therapy. Recent research has introduced exciting new theories regarding the pathogenesis of preeclampsia. Clinical and experimental evidence implicating the circulating antiangiogenic molecules soluble Fms-like tyrosine kinase-1 (sFLT-1) and soluble endoglin (sENG), as well as endothelin-1 and the angiotensin II receptor type I autoimmune antibody (AT-1AA), have been especially promising. This review collates evidence for a role of hypoxia and hypoxia-inducible factor-1alpha (HIF1A; referred to as HIF-1α throughout) in the pathogenesis of preeclampsia and discusses possible molecular links between hypoxia and the newly reported potential mediators of the disease's manifestations.


Assuntos
Hipóxia Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/etiologia , Animais , Antígenos CD/sangue , Antígenos CD/química , Antígenos CD/metabolismo , Autoanticorpos/análise , Endoglina , Endotelina-1/sangue , Endotelina-1/química , Endotelina-1/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Placenta/imunologia , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/imunologia , Pré-Eclâmpsia/metabolismo , Gravidez , Receptor Tipo 1 de Angiotensina/química , Receptores de Superfície Celular/sangue , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Solubilidade , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/sangue , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
20.
Life Sci ; 91(13-14): 607-12, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22406300

RESUMO

AIMS: Endothelin-1 (ET-1) and endothelin-2 (ET-2; Trp(6)Leu(7)ET-1) are expressed by different cell types, but are considered to display identical pharmacological properties on endothelin receptors. We studied agonist-dependent aspects of endothelin(A) (ET(A))-receptor function and the importance of amino acids 6 and 7 of ET-1 and ET-2 in this respect. MAIN METHODS: We used isolated rat mesenteric resistance arteries in wire myographs, in a setting that minimizes influences of endothelium and sensorimotor nerves, to study arterial smooth muscle ET(A)-receptor-mediated vasomotor responses, to ET-1, ET-2 and chimeras thereof (Trp(6)ET-1 and Leu(7)ET-1). KEY FINDINGS: ET-1 and ET-2 cause arterial contractions with comparable sensitivities and maximal responses. BQ123 (ET(A)-antagonist) reduces sensitivity to ET-1 more potently than that to ET-2 (pK(B): 7.1 ± 0.2 versus 5.6 ± 0.4). However, 1 µM BQ123 relaxes maximal contractile responses to ET-2 more markedly than those to ET-1. Leu(7)ET-1 is a contractile agonist with lower potency and similar maximal effect compared to ET-1 and greater sensitivity to BQ123 than ET-2. Up to 256 nM Trp(6)ET-1 did not cause contraction and did not antagonize arterial responses to ET-1. SIGNIFICANCE: Arterial smooth muscle ET(A)-receptor function displays agonist-dependent aspects. This involves roles of amino acids on position 6 and 7 of the endothelin sequence. Agonist-dependent pathologies may benefit from the design of specific, agonist-selective ET-receptor antagonists.


Assuntos
Endotelina-1/metabolismo , Endotelina-2/metabolismo , Artérias Mesentéricas/metabolismo , Receptor de Endotelina A/metabolismo , Sequência de Aminoácidos , Animais , Endotelina-1/química , Endotelina-2/química , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Ratos , Ratos Endogâmicos WKY , Receptor de Endotelina A/agonistas
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